Trp-p8 active compounds and therapeutic treatment methods

ABSTRACT

Compounds of the disclosure provide compositions, which are effective for prophylaxis and treatment of diseases or disorders, such as cell-proliferation, angiogenesis, or apoptosis mediated diseases. The disclosure encompasses compounds, analogs, prodrugs, metabolites, and pharmaceutically acceptable salts thereof, pharmaceutical compositions, and methods for prophylaxis and treatment of diseases and other maladies or conditions involving cancer, tumors, and like conditions. The disclosure also provides therapeutic methods including the administration of an effective amount of a compound of the disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/884,379,filed Jul. 2, 2004, which application claims the benefit of provisionalapplication Ser. No. 60/484,526, filed Jul. 2, 2003 and provisionalapplication Ser. No. 60/491,616, filed Jul. 31, 2003, which applicationsare incorporated herein by reference in their entirety.

BACKGROUND

Compounds which produce a physiological cool sensation when applied tothe skin are known, see for example, “New Compounds with the MentholCooling Effect,” H. R. Watson, et al., J. Soc. Cosmet. Chem., 1978, 29,185-200.

Wei, E. T., et al., J. Pharm. Pharmacol., 1983, 35(2), 110-112, describea compound named “icilin” for its cool-sensation producing properties,(also known as AG-3-5) or3-(2-Hydroxy-phenyl)-6-(3-nitro-phenyl)-3,4-dihydro-1H-pyrimidin-2-one,of the formula:

Still other compounds having cooling action have been recently reported,see H. Ottinger, et al., J. Agric. Food Chem., 2001, 49, 5383-5390.

U.S. Pat. No. 4,150,052, discloses menthane carboxamide compounds, forexample, of the formula IIa3-1:

that are reported to have a physiological cooling action on the skin.

U.S. Pat. No. 4,070,496, discloses certain phosphine oxide (R₁R₂R₃P═O)compounds and compositions that are reported to have a physiologicalcooling action on the skin.

U.S. Pat. No. 3,821,221, discloses certain tetrahydropyrimidine-2-onecompounds that are reported to have central nervous system activity asdepressants or stimulants.

Recently, certain TRP receptors have been shown to have a role inthermosensation, see D. D. McKemy, et al., “Identification of a ColdReceptor Reveals a General Role for TRP Channels in Thermosensation,”Nature, 2002, Mar. 7; 416(6876):52-8. For a recent review of “The TRPIon Channel Family,” see D. E. Clapham, et al., Nature Reviews,Neuroscience, 2001, 2, 387-396 <www.nature.com/reviews/neuro>. Okazawaet al Neuroscience letters (2004 Apr. 8), 359(1-2):33-6; Nealen et alJournal of neurophysiology (2003 July), 90(1):515-20; Thut et al.,Neuroscience (2003), 119(4):1071-83.

The gene Trp-p8 was discovered by screening a prostate-specificsubtracted cDNA library. The predicted protein has significant homologywith the transient receptor potential (Trp) family of Ca²⁺ channelproteins. Northern blot analysis indicates Trp-p8 expression withinnormal human tissues is mostly restricted to prostate epithelial cells.In situ hybridization analysis shows that Trp-p8 mRNA expression was atmoderate levels in normal prostate tissue and appears to be elevated inprostate cancer. Trp-p8 mRNA was also expressed in a number ofnon-prostatic primary tumors of breast, colon, lung, and skin origin,whereas transcripts encoding Trp-p8 were hardly detected or not detectedin the corresponding normal human tissues (Tsavaler, et al CancerResearch (2001), 61(9):3760-3769).

Immunotherapy of prostate carcinoma (PCa) largely depends on theidentification of suitable target antigens that are present in a highpercentage of prostate tumors. The putative calcium channel protein,Trp-p8, is associated with loss of Trp-p8 mRNA expression and asignificantly shorter time to PSA relapse-free survival. Theidentification of Trp-p8 is associated with prostate cancer outcome, andsuggests an integral role for this receptor in prostate carcinogensis.Immunogenic peptides derived from the prostate-specific proteintransient receptor potential-p8 (Trp-p8) that is recognized by cytotoxicT lymphocytes from PCa patients have been reported (Kiessling, et al(2003) Prostate 56(4):270-279; Henshall, et al Cancer Research (2003),63(14):4196-4203; Fuessel, et al International Journal of Oncology(2003), 23(1):221-228; US 2003108963 A1). Identification of therapeuticagents effective in the treatment of neoplastic, hyperplastic, and likediseases or conditions continues to be the subject of significantresearch efforts. Recent work indicates that certain therapeutic agentsin combination with certain antibody preparations can be effective intreating angiogenic related disorders, and like diseases or conditions,see for example, U.S. Pat. No. 6,582,959.

There is currently a need for therapeutic agents and methods that areuseful to treat diseases and conditions that are associated withregulation of the Trp-p8 receptor. There is also a need for therapeuticagents and treatment methods, which are specific and selective towardcancerous cells and have low cytotoxicity toward healthy cells.

There is also a need for therapeutic agents in combination withadditional chemotherapeutic agents, including, for example, antibodypreparations, and combination therapeutic treatment methods thereof,that are useful to treat diseases and conditions that are associatedwith regulation of the Trp-p8 receptor.

SUMMARY

It has now been discovered certain compounds, including some known toproduce a physiological cool sensation (“cool-genic”), such as the abovementioned tetrahydropyrimidine-2-ones, phosphine oxides, menthanecarboxamide compounds, alkyl substituted alkyl amide compounds, andalpha-keto enamines, exhibit useful biological activity, such asanti-tumor activity.

Accordingly, the present disclosure provides, in exemplary embodiments,compounds which activate the Trp-p8 receptor. In embodiments, thepresent disclosure provides compounds and treatment methods, which causeincreased calcium ion flow into cancerous cells (i.e., “flux activating”or “flux promoting” compounds). In embodiments, the present disclosureprovides compounds, pharmaceutical compositions, and treatment methods,which inhibit or kill cancerous cells, for example, by causingapoptosis.

In embodiments, the present disclosure also provides:

a pharmaceutical composition comprising a compound of the disclosure anda pharmaceutically acceptable excipient (the composition preferablycomprises a therapeutically effective amount of the compound or salt);

a pharmaceutical composition for use in the treatment of tumors, whichcomprises a compound of the disclosure, and combinations thereof, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

a method for treating a disease or condition in a mammal (e.g., a human)wherein a Trp-p8 receptor is implicated and modulation of receptorfunction is desired comprising administering an effective modulatoryamount of a compound of the disclosure;

a method for treating or preventing a disease or Trp-p8 receptor relatedcondition (e.g., tumors) in a mammal comprising administering atherapeutically effective amount of a compound of the disclosure;

a compound of the disclosure for use in medical diagnosis or therapy(e.g., the treatment or prevention of Trp-p8 receptor related disease orcondition such as tumors);

the use of a compound of the disclosure to prepare a medicament usefulfor treating or preventing a disease or Trp-p8 receptor relatedcondition (e.g., the treatment or prevention of Trp-p8 receptor relateddisease or condition such as tumors);

a method of treating cancer, for example, tumors, comprisingadministering to a mammal in need of such treatment, an effective amountof a compound of the disclosure;

a method for modulating Trp-p8 receptor function comprisingadministering an effective modulatory amount of a compound of thedisclosure; and

a method for modulating Trp-p8 receptor function comprising contactingan Trp-p8 receptor with an effective modulatory amount of a compound ofthe disclosure.

We have also discovered certain compounds of the disclosure, forexample, those compounds known to produce a physiological cool sensation(“cool-genic”) and structurally related compounds, such as the abovementioned tetrahydropyrimidine-2-ones, phosphine oxides, menthanecarboxamide compounds, alkyl substituted alkyl amide compounds,alpha-keto enamines, and like compounds, when used in combination withadditional chemotherapeutic agents, including, for example, antibodies,are capable of useful biological activity, such as anti-tumor activity.

Accordingly, the present disclosure provides in embodiments, therapeuticcombinations of a compound of the disclosure (which can activate theTrp-p8 receptor), and additional chemotherapeutic agents, including, forexample, an antibody. In embodiments, the present disclosure providessuch therapeutic combinations which can be effective in the treatment ofcancerous cells. In embodiments, the present disclosure provides suchtherapeutic combinations and treatment methods thereof, which can beeffective in inhibiting or killing cancerous cells, for example, bycausing apoptosis, inhibiting angiogenesis, or both.

In embodiments, the present disclosure also provides:

a composition comprising a compound of the disclosure in combinationwith an antibody and a pharmaceutically acceptable carrier;

a pharmaceutical composition comprising therapeutic combinations of acompound of the disclosure and an antibody, and a pharmaceuticallyacceptable excipient (the composition preferably comprises atherapeutically effective amount of the compound or salt and atherapeutically effective amount of at least one additionalchemotherapeutic agent, for example, an anti-angiogenic antibody);

a pharmaceutical composition for use in the treatment of tumors, whichcomprises an effective amount of a compound of the disclosure, or apharmaceutically acceptable salt thereof, in combination with aneffective anti-tumor amount of at least one additional chemotherapeuticagent, for example, an anti-angiogenic antibody, and a pharmaceuticallyacceptable carrier;

a method for treating a disease or condition in a mammal (e.g., a human)wherein a Trp-p8 receptor is implicated (e.g., wherein the disease orcondition is characterized by over-expression Trp-p8 receptors) andmodulation of receptor function is desired comprising administering aneffective modulatory amount of a compound of the disclosure incombination with an effective anti-cancer amount of at least oneadditional chemotherapeutic agent, for example, an anti-angiogenicantibody;

a method for treating or preventing a disease or Trp-p8 receptor relatedcondition (e.g., tumors) in a mammal comprising administering atherapeutically effective amount of a combination of compound of thedisclosure and at least one additional chemotherapeutic agent, forexample, an anti-angiogenic antibody;

a compound of the disclosure in combination with at least one additionalchemotherapeutic agent, for example, an effective amount of ananti-angiogenic antibody for use in medical diagnosis or therapy (e.g.,the treatment or prevention of Trp-p8 receptor related disease orcondition such as tumors);

the use of a compound of the disclosure in combination with an effectiveamount of at least one additional chemotherapeutic agent, for example,an anti-angiogenic antibody to prepare a medicament useful for treatingor preventing a disease or Trp-p8 receptor related condition (e.g., thetreatment or prevention of Trp-p8 receptor related disease or conditionsuch as tumors);

a method of treating cancer, for example, tumors, comprisingadministering to a mammal in need of such treatment, an effective amountof a compound of the disclosure in combination with an effective amountof at least one additional chemotherapeutic agent, for example, ananti-angiogenic antibody;

a method for modulating Trp-p8 receptor function comprisingadministering an effective modulatory amount of a compound of thedisclosure in combination with an effective amount of at least oneadditional chemotherapeutic agent, for example, an anti-angiogenicantibody; and

a method for modulating Trp-p8 receptor function comprising contactingan Trp-p8 receptor with an effective modulatory amount of a compound ofthe disclosure in combination with an effective amount of at least oneadditional chemotherapeutic agent, for example, an anti-angiogenicantibody.

We have also discovered certain other compounds, such as certainmenthane carboxamide compounds described below, characterized in thatthey also exhibit useful biological activity, such as cell killinganti-tumor activity.

Accordingly, in embodiments, the present disclosure also providescompounds of the formula IIa:

wherein

R₁ is H, or (C₁-C₆)alkyl;

R₂ is phenyl or a substituted phenyl of the formula (-PhR₃R₄R₅R₆R₇)

where

R₃, R₄, R₆, and R₇ are each independently —H, (C₁-C₆)alkyl,(C₁-C₆)alkoxyl, or halo;

R₅ is halo, (C₁-C₆)alkyl, (C₃-C₁₂)cycloalkyl, (C₁-C₆)alkoxyl,—C(═O)(C₁-C₆)alkyl or (C₁-C₇)alkanoyl;

or R₅ is —NR₈R₉, where R₈ and R₉ are each independently —H,(C₁-C₆)alkyl, or R₈ and R₉ together with the nitrogen to which they areattached form a morpholino, pyrrolidino, piperidino, piperzino,indolino, benzimidazolino, azetidino, aziridino, azepino, 1,4-oxazino,or thiomorpholino ring;

or R₄ and R₅ together with the phenyl to which they are attached form aring having 4 to 7 atoms and the ring having from 1 to 3 unsaturations;and

stereoisomeric forms, mixtures of stereoisomeric forms;

or a pharmaceutically acceptable salt thereof,

provided that when R₃, R₄, R₆, and R₇ of -PhR₃R₄R₅R₆R₇ are —H, R₅ isother than —CH₃, —OCH₃, —OH, —F, or —NO₂; and

provided that R₂ is other than 3-hydroxy-4-methyl-phenyl; and furtherprovided

that R₂ is other than 2-hydroxy-naphthyl, or pyridyl.

In embodiments, the present disclosure also provides a compound of theabove mentioned formula IIa, which is characterized in that the compoundis effective in killing cells expressing TRP-p8 but where the calciumion flux of the expressing cell need not be substantially changed by thepresence of the compound.

In embodiments, the present disclosure also provides:

a method for killing target cells which express Trp-p8 but withoutsubstantially changing the calcium flux characteristics of the targetcells (e.g., tumors) in a mammal comprising administering atherapeutically effective amount of a compound of the disclosure of theformula IIa;

a method for treating a disease or condition in a mammal (e.g., a human)comprising administering an effective amount of a compound of thedisclosure of the formula IIa, which compound is cytotoxic with respectto the Trp-p8 receptor and increased calcium ion flux;

a method for treating or preventing a disease or Trp-p8 receptor relatedcondition (e.g., tumors) in a mammal comprising administering atherapeutically effective amount of a compound of the disclosure of theformula IIa, which compound is cytotoxic with respect to the Trp-p8receptor; and

any of the above methods for killing cells, for treating a disease orcondition, or treating or preventing a disease comprising administeringa therapeutically effective amount of a compound of the disclosure ofthe formula IIa in combination with another compound of the disclosure,an anti-angiogenic antibody, or mixtures thereof.

These and other embodiments are illustrated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D illustrate the effectiveness of selected cool-genic compoundsof the present disclosure in killing human cells expressing Trp-p8compared to the relative insensitivity of human cells not expressingTrp-p8.

FIG. 2 illustrates the relative growth rates for cloned cancerous celllines (PC3/Trp-p8) which express Trp-p8 compared to a control cell line(PC3-Neo) which does not express Trp-p8.

DETAILED DESCRIPTION

The present disclosure provides the abovementioned pharmaceuticalcompositions and methods of treatment. The present disclosure alsoprovides the abovementioned compounds of the disclosure of the formulaIIa, pharmaceutical compositions including a compound of the disclosureof the formula IIa, and methods of treatment therewith, and whichcompounds, and pharmaceutical compositions, are cytotoxic to Trp-p8expressing cells.

Examples of cool-genic compounds are, for example, a compound of theformulas (I-XIII):

wherein R₁ and R₂ are each independently H, alkyl, Het, or aryl, or asdisclosed in U.S. Pat. No. 3,821,221;

wherein R₁ and R₂ are each independently H, alkyl, or aryl, or asdisclosed, for example, in U.S. Pat. No. 4,150,052, and J. Soc. Cosmet.Chem., 1978, 29, 185-200;

wherein R₁, R₂, and R₃ are each independently H, alkyl, or aryl, or asdisclosed, for example, in J. Soc. Cosmet. Chem., 1978, 29, 185-200 (andreferences cited therein such as reference 1);

wherein R₁, R₂, and R₃ are each independently linear or branched alkylor cycloalkyl, or as disclosed, for example, in U.S. Pat. No. 4,070,496;

wherein R₃ is —OH, —S(O)R₁, —P(═O)R₁R₂, —CO₂H, —C(═O)NH₂,—OC(═O)—CH(OH)—CH₃, —C(═O)OC_(n)H_(2m)—OH, where n is 1-4,—NR₁—C(═O)NR₁R₂, —SO₂R₁, —SO₂NR₁R₂, —SONR₁R₂, and where R₁ and R₂ areeach independently H, alkyl, or aryl, and R₄ is H, or R₃ and R₄ takentogether with the carbon atom to which they are attached is a 5-memberketal ring optionally having an hydroxymethyl substituent of the formula—OCH₂—CH(CH₂—OH)—O—, or as disclosed, for example, in J. Soc. Cosmet.Chem., 1978, 29, 185-200;

a core selected from the (—X) substituted cyclic or branchedhydrocarbons of group VI:

wherein X is an N-alkyl carboxamide, —C(═O)NR₁R₂, where R₁ and R₂ areeach independently H, alkyl, or aryl, or R₁ and R₂ taken together withthe nitrogen atom to which they are attached is a 5- or 6-memberedsaturated or unsaturated heterocyclic (Het) ring which is optionallysubstituted with an oxygen (—O—) ring heteroatom, or as disclosed, forexample, in J. Soc. Cosmet. Chem., 1978, 29, 185-200;

wherein X is an N-alkyl carboxamide, —C(═O)NHR₁, where R₁ is alkyl orsubstituted alkyl, such as in —C(═O)NHEt or —C(═O)NHCH₂CO₂Et, or alkylsulfone, such as in —SO₂Et, or as disclosed, for example, in J. Soc.Cosmet. Chem., 1978, 29, 185-200;

alkyl substituted urea compounds, for example, of the formula VIII:

or as disclosed, for example, in J. Soc. Cosmet. Chem., 1978, 29,185-200;

wherein R₁ and R₂ are each independently alkyl, orR₁ and R₂ taken together with the nitrogen atom to which they areattached is a 5- or 6-membered saturated or unsaturated heterocyclic(Het) ring which is optionally substituted with an oxygen (—O—) ringheteroatom, such as a morpholino-ring, and the ring can be optionallysubstituted with an alpha-CO₂H or an alpha-CO₂CH₃ substituent, andR₃, R₄, and R₅ are each independently H or alkyl, or as disclosed, forexample, in J. Agric. Food Chem., 2001, 49, 5383-5390;

wherein R₁ and R₂ are each independently alkyl, or R₁ and R₂ takentogether with the nitrogen atom to which they are attached is a 5- or6-membered saturated or unsaturated heterocyclic (Het) ring, and R₃, R₄,R₅, and R₆ are each independently H or alkyl, or as disclosed, forexample, in J. Agric. Food Chem., 2001, 49, 5383-5390;

wherein R₁ and R₂ are each independently alkyl, orR₁ and R₂ taken together with the nitrogen atom to which they areattached is a 5- or 6-membered saturated or unsaturated heterocyclic(Het) ring, andR₃ and R₄ are each independently H or alkyl, or as disclosed, forexample, in J. Agric. Food Chem., 2001, 49, 5383-5390;

wherein R₁ and R₂ are each independently alkyl, orR₁ and R₂ taken together form a 5- or 6-membered saturated orunsaturated ring, andR₃ and R₄ are each independently H or alkyl, or as disclosed, forexample, in J. Agric. Food Chem., 2001, 49, 5383-5390; or

wherein R₁ to R₅ are each independently H, alkyl, or aryl, R₆ is —OH,—S(O)R₈, —P(═O)R₈R₉, —CO₂H, —C(═O)NH₂, —OC(═O)—CH(OH)—CH₃,—C(═O)OC—H₂—OH, where n is 1-4, —NR₈—C(═O)NR₈R₉, —SO₂R₈, —SO₂NR₈R₉, or—SONR₈R₉, and where the R₈ and R₉ of R₆ are each independently H, alkyl,or aryl, and R₇ is H, or R₆ and R₇ taken together with the carbon atomto which they are attached is a 5-member ketal ring, having anhydroxymethyl substituent, of the formula —OCH₂—CH(CH₂—OH)—O—;or a pharmaceutically acceptable salt thereof.

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc., denote bothstraight and branched groups; but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical, a branchedchain isomer such as “isopropyl” being specifically referred to.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (1-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃),1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃),3-pentyl (—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃),3-methyl-2-butyl (—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂),2-methyl-1-butyl (—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃),2-hexyl (—CH(CH₃)CH₂CH₂CH₂CH₃)_(,) 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

“Aryl” denotes a phenyl radical or an ortho-fused bicyclic carbocyclicradical having about nine to twenty ring atoms in which at least onering is aromatic. Aryl (Ar) can include substituted aryls, such as aphenyl radical having from 1 to 5 substituents, for example, alkyl,alkoxy, and like substituents, and which substituents are consistentwith the compounds and inclusive of the substituents disclosed in theabove mentioned patents or publications for compounds of the formulas(I-XIII).

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multi-specific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity. An antibody is a proteingenerated by the immune system that is capable of recognizing andbinding to a specific antigen. Described in terms of its structure, anantibody is a Y-shaped protein consisting of four amino acid chains, twoheavy and two light. In a simplified model sufficient for this appeal,each antibody has primarily two regions: a variable region and aconstant region. The variable region, located on the ends of the arms ofthe Y, binds to and interacts with the target antigen. This variableregion includes a complementary determining region (CDR) that recognizesand binds to a specific binding site on a particular antigen. Theconstant region, located on the tail of the Y, is recognized by andinteracts with the immune system (Janeway, C., Travers, P., Walport, M.,Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).A target antigen generally has numerous binding sites, also calledepitopes, recognized by CDRs on multiple antibodies. Each antibody thatspecifically binds to a different epitope has a different structure.Thus, one antigen may have more than one corresponding antibody.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628 andMarks et al (1991) J. Mol. Biol., 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc) and human constant region sequences.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); antibodies that have an anti-cancer effect, particularlythose that bind to VEGF-1, VEGF-2, VEGF-3, EGF-R, HER-2, CD20, and thelike, and pharmaceutically acceptable salts, acids or derivatives of anyof the above.

“Het” is a four- (4), five- (5), six- (6), or seven- (7) memberedsaturated or unsaturated heterocyclic ring having 1, 2, 3, or 4heteroatoms selected from the group consisting of oxy, thio, sulfinyl,sulfonyl, and nitrogen, which ring is optionally fused to a benzenering. Het includes “heteroaryl,” which encompasses a radical attachedvia a ring carbon of a monocyclic aromatic ring containing five or sixring atoms consisting of carbon and 1, 2, 3, or 4 heteroatoms eachselected from the group consisting of non-peroxide oxy, thio, and N(X)wherein X is absent or is H, O, C₁₋₄alkyl, phenyl or benzyl, as well asa radical of an ortho-fused bicyclic heterocycle of about eight to tenring atoms derived therefrom, particularly a benz-derivative or onederived by fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

“Treat,” “treatment,” “treating,” and like terms refer, in embodiments,to lessen, to eliminate, to inhibit, to improve, to alter, or to preventa disease or condition, for example by administration of an effectiveamount of a compound of the present disclosure, or by administration ofan effective amount of a compound of the disclosure in combination withan effective amount of an additional chemotherapeutic agent ananti-angiogenic antibody, and can refer to curative therapy,prophylactic therapy, and preventative therapy.

“Modulate,” “modulation,” “modulating,” “modulatory,” and like termsrefer to, in embodiments, the ability of an effective amount of acompound of the present disclosure to, selectively and at relatively lowdose levels, adjust or alter the multi-valent ion (such as calcium ions)permeability of cancerous cells, such as those cells expressing Trp-p8,and in contrast to the relative insensitivity of other cells, forexample, healthy or non-cancerous cells and cells not expressing Trp-p8.

“Cancer treatment,” “treating cancer,” and like terms, for purposes ofthis disclosure refer, in embodiments, to a method of treating cancerwhich includes contacting cancer cells with a compound of the disclosurein order to achieve an inhibition of cancer cell growth, a killing ofcancer cells, increased patient survival time, or a combination thereof,or contacting cancer cells with a compound of the disclosure incombination with an anti-angiogenic antibody in order to achieve aninhibition of cancer cell growth, a killing of cancer cells, increasedpatient survival time, an anti-angiogenic effect, or a combinationthereof. Treatment of cancer, by the method of the disclosure, alsoincludes contacting the cells with a compound of the disclosure toactivate Trp-p8 receptors in cancerous cells, such as tumors, to causeelevated flux levels of multi-valent ions into the cells to cause cellstasis or cell death. Cancer can include diseases in which abnormalcells divide without control. Cancer cells can also invade nearby tissueand can spread through the bloodstream and lymphatic system to otherparts of the body. The major categories of cancers are carcinomas,sarcomas, leukemias, and lymphomas. Within these major categories arenumerous subgroups that generally describe the organ in which the canceroriginates, such as adenocarcinoma of the stomach or oat cell carcinomaof the lung.

“Tumor” refers to, for example, an abnormal benign or malignant mass oftissue that may not be inflammatory, arises from cells of pre-existenttissue, and may possesses no physiological function. Benign tumors,include for example, cysts, warts, moles, and polyps, and generally donot spread to other parts of the body. Malignant tumors are typicallycomposed of cells that grow rapidly, have other abnormal properties thatdistinguish them from normal cells, and often invade other normaltissues.

“Vascular endothelial cell growth factor,” or “VEGF,” refers to amammalian growth factor as defined, for example, in U.S. Pat. No.5,332,671. The biological activity of native VEGF is shared by anyanalog or variant thereof that promotes selective growth of vascularendothelial cells but not of bovine corneal endothelial cells, lensepithelial cells, adrenal cortex cells, BHK-21 fibroblasts, orkeratinocytes.

“Angiogenic disorder” or “angiogenic defect” refers to an abnormalcondition that requires treatment with an agent that inhibitsangiogenesis, e.g., an angiostatic compound or composition such as acombination of a compound of the disclosure and an anti-angiogenicantibody. Such disorders include, for example, types of cancer such asvascular tumors, e.g., hemangioma (capillary and cavernous), glomustumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, and tumor angiogenesis.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The cool-genic compounds are suitable for use in mammals. As usedherein, “mammals” means any class of higher vertebrates that nourishtheir young with milk secreted by mammary glands, including, forexample, humans, horses, cows, pigs, sheep, dogs, rabbits, and monkeys.

“Apoptotic cell death,” “programmed cell death,” “apoptosis” and liketerms refer to any cell death that may result from the complex cascadeof cellular events that occur at specific stages of cellulardifferentiation and in response to specific stimuli. Apoptotic celldeath can be characterized by condensation of the cytoplasm and nucleusof dying cells. Apoptosis is an active process requiring new proteinsynthesis. Typically, the process requires ATP, involves new RNA andprotein synthesis, and culminates in the activation of endogenousendonucleases that degrade the DNA of the cell, thereby destroying thegenetic template required for cellular homeostasis. Apoptosis isobserved in controlled deletion of cells during metamorphosis,differentiation, and general cell turnover, and appears normally to beregulated by receptor-coupled events. For these reasons, apoptosis hasbeen called “programmed cell death” or “cell suicide.” While every celllikely has a genetic program to commit suicide, it is usuallysuppressed. Under normal circumstances, only those cells no longerrequired by the organism activate this self-destruction program.

“Therapeutically effective amount” means, in embodiments, a dose of acompound of the disclosure, or a dose of a combination of a compound ofthe disclosure and another chemotherapeutic agent, with or without anexcipient, that inhibits, reduces or eliminates tumor growth, in vivo,in vitro, or both, by for example, activating Trp-p8, stimulatingapoptosis, or both. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques.

In one embodiment of the present disclosure, the compounds of thedisclosure are used in combination therapy, for example, with otherantibody therapeutic agents. In an embodiment, compounds of the presentdisclosure are used in combination with known cancer treatingantibodies. See generally, for example: PCT/US02/19592; PCT/US01/20118;PCT/US01/25464; PCT/US01/26626; PCT/US02/28859; PCT/US02/41798;PCT/US02/12206; PCT/US03/11148; PCT/US02/12619; and PCT/US02/33050. Inanother embodiment, compounds of the disclosure are used in combinationwith an anti-VEGF antibody and like antibodies including human,non-human, murine, hybrid, and chimeric forms. See for example U.S. Pat.No. 6,582,959 (VEGF) and U.S. Patent Application No. 2002/0122797 A1(human VEGF).

In embodiments of the present disclosure, compounds of the disclosurecan be used in combination with other therapeutic agents, such as theantibodies noted above, to treat immunological diseases or conditions,for example, involving immune responsive cells such as B-cells (Blymphocytes), T-cells (T lymphocytes), accessory cells (macrophages andother antigen-presenting cells), killer cells (NK and K cells), mastcells, and like cells.

In a preferred embodiment, compounds useful in the present disclosureinclude a non-radio labeled compound for treatment of non-centralnervous system cells or diseases. In embodiments, compounds of thepresent disclosure do not contain a radio-label and are notradio-active. The unlabeled compounds of the present disclosure can beused to kill cancer cells as illustrated herein, for example, cellsexpressing Trp-p8, such as prostate cancer cells and liver cancer cells.

A “subject” for the purposes of the present disclosure includes bothhumans and other animals, particularly mammals. Thus, the methods areapplicable to both human therapy and veterinary applications. In apreferred embodiment the subject is a mammal, and in the most preferredembodiment the subject is human.

“Improved therapeutic outcome” or “decrease in the number of tumorcells” or “decreased tumor size” means a 50% decrease, preferably an 80%decrease, more preferably a 90% decrease, and even more preferably a100% decrease in the tumor size or volume, a decrease in the number ofdetectable circulating cancer cells in the blood, affected tissue, ororgan as determined by examination of a patient, samples taken from apatient prior to and following treatment, or both.

“Compound,” “molecule,” “polypeptide,” and like terms includesynthetically prepared compounds, genetically engineered compounds(e.g., recombinant DNA expressed proteins), naturally occurringcompounds, and those produced in vivo after administration of adifferent compound. The in vivo effects of administered compoundsdescribed herein may not be exerted by those compounds as such, but byone or more degradation products, such as a metabolite, conjugate,clathrate, ion complex, chelate, hydrate, solvate, or like biologicaltransformations or combinations, of the administered compound(s) ormolecule(s). “Pharmaceutically acceptable salts” can also include, inaddition to those illustrated herein, a subclass of salts present orformed in vivo.

The present disclosure provides compounds that bind to certain receptorsin the TRP (transient receptor potential) ion channel family. Moreparticularly the present disclosure provides compounds that specificallybind to the subgroup of long TRP (or TRPM) channels, and mostparticularly to compounds that specifically bind to the TRP channelcalled “Trp-p8” (or TRP-M8). The Trp-p8 receptors are typically presentat elevated levels in cancers, such as prostate cancer. The compounds ofthe disclosure are endowed with Trp-p8 receptor activating activity.Activation of the Trp-p8 receptor causes increased calcium ion flow intocancerous cells and ultimately cell death. Compounds of the presentdisclosure are useful for, but not limited to, the treatment of cellproliferation diseases or disorders, and stimulating apoptosis. It isalso well known in the art and as illustrated herein how to determineTrp-p8 receptor activity, for example, using the standard testsdescribed herein, or using other similar tests.

It will be appreciated by those skilled in the art that compounds of thedisclosure having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present disclosureencompasses any racemic, optically-active, polymorphic, tautomeric, orstereoisomeric form, or mixture thereof, of a compound of thedisclosure, which possesses the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase). In particular, it is understood that compounds of thedisclosure, such as of formulas (I-XIII), can contain chiral centers,for example, in the R₁ substituents of formula (I), the R₃ substituentsof formula (V), and in the R₁ to R₆ substituents of formula (XIII). Itis also understood that compounds of the disclosure, such as formula(I), can exist in the “enol” form or the corresponding tautomeric “keto”form, and that all such tautomers are included as compounds within thescope of the present disclosure.

The carbon atom content of various hydrocarbon-containing moieties isindicated by a prefix designating a lower and upper number of carbonatoms in the moiety, i.e., the prefix C_(i-j) indicates a moiety of theinteger “i” to the integer “j” carbon atoms, inclusive. Thus, forexample, C₁₋₆alkyl or (C₁-C₆)alkyl refers to alkyl of one to six carbonatoms, inclusive.

The compounds of the present disclosure are generally named according tothe IUPAC nomenclature system. Abbreviations, which are well known toone of ordinary skill in the art, may be used (e.g., “Ph” for phenyl,“Me” for methyl, “Et” for ethyl, “h” for hour or hours and “rt” for roomtemperature).

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents. The compounds of the disclosure include compounds offormula (I-XIII) having any combination of the values, specific values,more specific values, and preferred values described herein.

Specifically, aryl can be phenyl, naphthyl, anthracenyl, phenanthrenyl,fluorenyl, tetrahydronaphthyl, or indanyl.

Specifically, alkyl such as (C₁₋₆)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, 3-pentyl,hexyl, or heptyl; (C₂-C₆)alkyl, can be ethyl, propyl, isopropyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, 3-pentyl, or hexyl;(C₃₋₁₂)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, bicyclic, or multi-cyclicsubstituents, such as of the formulas

C₁₋₆alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy,iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; —C(═O)alkyl or(C_(2—)C₇)alkanoyl can be acetyl, propanoyl, butanoyl, pentanoyl,4-methylpentanoyl, hexanoyl, or heptanoyl; aryl can be phenyl, indenyl,or naphthyl; Het can be pyrrolidinyl, piperidinyl, morpholinyl,thiomorpholinyl, or heteroaryl; and heteroaryl can be furyl, imidazolyl,triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl,pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide),thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or itsN-oxide) or quinolyl (or its N-oxide).

A specific value for Het is a five- (5), six- (6), or seven- (7)membered saturated or unsaturated ring containing 1, 2, 3, or 4heteroatoms, for example, non-peroxide oxy, thio, sulfinyl, sulfonyl,and nitrogen; as well as a radical of an ortho-fused bicyclicheterocycle of about eight to twelve ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, tetramethylene or another monocyclic Het diradicalthereto.

A specific compound of formula (I) is a compound of the Formula (A)

A specific compound of formula (II) is a compound of the Formula (B)

Another specific compound of formula (II) is a compound of the Formula(C) showing a preferred stereochemistry:

Another specific compound of formula (II) is a compound of the Formula(D):

A specific compound of formula (III) is a compound of the Formula (E):

A specific compound of formula (IV) is a compound of the Formula (F):

A specific compound of formula (V) is a compound of the Formula (G):

A specific compound of formula (VI) is a compound of the Formula (H):

A specific compound of formula (VII) is a compound of the Formula (I′):

A specific compound of formula (IX) is a compound of the Formula (J):

A specific compound of formula (X) is a compound of the Formula (K):

A specific compound of formula (XI) is a compound of the Formula (L):

A specific compound of formula (XII) is a compound of the Formula (M):

A specific compound of formula (XIII) is a compound of the Formula (N):

It is understood that the abovementioned specific compounds of theFormulas (A-N), and all other compounds of the disclosure, can be orinclude a pharmaceutically acceptable salt thereof.

A specific compound is3-(2-Hydroxy-phenyl)-6-(3-nitro-phenyl)-3,4-dihydro-1H-pyrimidin-2-one;or a pharmaceutically acceptable salt thereof.

Another specific compound is 2-Isopropyl-5-methyl-cyclohexanecarboxylicacid (4-methoxy-phenyl)-amide; or a pharmaceutically acceptable saltthereof.

Another specific compound is 2-Isopropyl-2,3, —N-trimethyl-butyramide;or a pharmaceutically acceptable salt thereof.

Another specific compound is 1-(sec-Butyl-isobutyl-phosphinoyl)-heptane;or a pharmaceutically acceptable salt thereof.

Another specific compound is 2-Isopropyl-5-methyl-cyclohexanol; or apharmaceutically acceptable salt thereof

A specific compound of formula IIa is a compound of the formula Ha1:

or a pharmaceutically acceptable salt thereof; wherein R₁, R₃, R₄, R₅,R₆, and R₇ are as defined herein.

Other specific compounds of formula IIa are individual compounds of theformulas IIa2-IIa12, or a pharmaceutically acceptable salt thereof;wherein the R substituents are as defined herein:

Specific compounds of formula IIa include:

-   2-Isopropyl-5-methyl-cyclohexanecarboxylic acid    (4-morpholin-4-yl-phenyl)-amide, or a pharmaceutically acceptable    salt thereof;-   2-Isopropyl-5-methyl-cyclohexanecarboxylic acid    (3-chloro-4-methoxy-phenyl)-amide, or a pharmaceutically acceptable    salt thereof;-   2-Isopropyl-5-methyl-cyclohexanecarboxylic acid    (4-sec-butyl-phenyl)-amide,-   or a pharmaceutically acceptable salt thereof;-   2-Isopropyl-5-methyl-cyclohexanecarboxylic acid indan-5-ylamide, or    a pharmaceutically acceptable salt thereof;-   2-Isopropyl-5-methyl-cyclohexanecarboxylic acid    (4-tert-butyl-phenyl)-amide, or a pharmaceutically acceptable salt    thereof;-   2-Isopropyl-5-methyl-cyclohexanecarboxylic acid    (4-propyl-phenyl)-amide, or a pharmaceutically acceptable salt    thereof and 2-Isopropyl-5-methyl-cyclohexanecarboxylic acid    (4-isopropyl-3-methyl-phenyl)-amide,

or a pharmaceutically acceptable salt thereof.

Preparative procedures, characterization, cool-genic properties, odorproperties, structure-coolgenic activity relationships, design rules,and like information for compounds of the formulas (I-XIII) and for theabove mentioned specific compounds of formulas A-N are reported in thecorresponding above mentioned publications or patents.

Compounds of the disclosure, such as compounds of formulas B, C, D or Ncan be prepared as illustrated, for example, in the scheme below, byprocedures analogous thereto, by procedures in the above mentionedpublications or patents, or by procedures which are known or would bereadily evident to one of ordinary skill in the art. All of thevariables used in the schemes are as defined below or elsewhere herein.Scheme 1 illustrates the preparation of representative compounds of thedisclosure, such as amide compound 3 (IIa-4-1). Chloro compound 1 wascarboxylated via a Grignard intermediate to afford carboxylic acid 2 andthe acid 2 was converted to the amide 3 and as described in Example 1herein.

An alternative preparative procedure for preparing compound 3 (IIa-4-1)and related amide compounds uses the corresponding acid chloride of theabove mentioned carboxylic acid compound 2. The acid chloride ofcarboxylic acid compound 2 (readily prepared by reaction with SOCl₂ andlike reagents) can be reacted with a variety of primary or secondaryamine compounds to form the corresponding amides analogous to amide 3.Other compounds related to amide 3 or formula II, were similarlyprepared and as illustrated and described herein.

For a synthetic procedure for making menthanecarboxylic acid compound 2(a starting material for IIa-4-1), see D. G. Rowsell, Wilkinson SwordLtd., U.K. Patent (1975) in the listing mentioned below. For anothersynthetic procedure for making the menthanecarboxylic acid, see D.Cunningham, et. al., J. Chem. Soc., Perkin Trans. I, 2002, 2692-2698.

For additional preparative details for making cool-genic compoundsmentioned in J. Soc. Cosmet. Chem., 1978, 29, 185-200, see on page 199,reference 1, listing of 17 U.K. Patents. Other conditions suitable forformation of the compounds of the disclosure from a variety ofintermediates as illustrated herein are well known to the art. Forexample, see Feiser and Feiser, “Reagents for Organic Synthesis,” Vol.1, 1967; March, J. “Advanced Organic Chemistry,” 4^(th) ed., John Wiley& Sons, 1992; House, H. O., “Modern Synthetic Reactions”, 2^(nd) ed., W.A. Benjamin, New York, 1972; and Larock, R. C., “Comprehensive OrganicTransformations,” 2^(nd) ed., Wiley-VCH Publishers, New York, 1999.

The starting materials employed in the synthetic methods describedherein are commercially available, have been reported in the scientificliterature, or can be prepared from readily available starting materialsusing procedures known in the field. It may be desirable to optionallyuse a protecting group during all or portions of the above described oralternative synthetic procedures. Such protecting groups and methods fortheir introduction and removal are well known in the art. See Greene, T.W.; Wutz, P. G. M. “Protecting Groups In Organic Synthesis” 2^(nd) ed.,New York, John Wiley & Sons, Inc., 1991.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound of thedisclosure as a salt may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids,which form a physiological acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitableinorganic salts may also be formed, including hydrochloride,hydrobromide, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example, by reacting asufficiently basic compound such as an amine with a suitable acidaffording a physiologically acceptable anion. Alkali metals, forexample, sodium, potassium or lithium, or alkaline earth metal salts,for example, calcium, of carboxylic acids can also be made.

Compounds of the present disclosure can conveniently be administered ina pharmaceutical composition containing the compound in combination witha suitable excipient, the composition being useful in treating tumors.Pharmaceutical compositions containing a compound appropriate foranti-tumor use are prepared by methods and contain excipients well knownin the art. A generally recognized compendium of such methods andingredients is Remington's Pharmaceutical Sciences by E. W. Martin (MarkPubl. Co., 15^(th) ed., 1975). The compounds and compositions of thepresent disclosure can be administered parenterally, for example, byintravenous, intraperitoneal or intramuscular injection, orally, orrectally, depending on, for example, the disposition or dissemination ofthe tumor cells.

In embodiments, the antibodies included within the scope of thedisclosure include hybrid and recombinant antibodies (e.g., “humanized”and “human” antibodies) regardless of species of origin orimmunoglobulin class or subclass designation, as well as antibodyfragments (for example, Fab, F(ab′)₂, and F_(v)), See U.S. Pat. No.4,816,567; Mage and Lamoyi, in Monoclonal Antibody Production Techniquesand Applications, 79-97, Marcel Dekker, Inc., New York, (1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from such a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies of the disclosure may be made using the hybridoma methodfirst described by Kohler & Milstein, Nature, 256:495 (1975), or may bemade by recombinant DNA methods. See U.S. Pat. No. 4,816,567. Otherknown methods of antibody production are described, for example, inGoding, Monoclonal Antibodies: Principles and Practice, 59-103, AcademicPress (1986); Kozbor, J. Immunol., 133:3001 (1984). Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, 51-63,Marcel Dekker, Inc., New York (1987).

Various methods have been employed to produce monoclonal antibodies(MAbs). Hybridoma technology, which refers to a cloned cell line thatproduces a single type of antibody, uses the cells of various species,including mice (murine), hamsters, rats, and humans. Another method toprepare MAbs uses genetic engineering including recombinant DNAtechniques. Monoclonal antibodies made from these techniques include,among others, chimeric antibodies and humanized antibodies. A chimericantibody combines DNA encoding regions from more than one type ofspecies. For example, a chimeric antibody may derive the variable regionfrom a mouse and the constant region from a human. A humanized antibodycomes predominantly from a human, even though it contains nonhumanportions. Like a chimeric antibody, a humanized antibody may contain acompletely human constant region. But unlike a chimeric antibody, thevariable region may be partially derived from a human. The nonhuman,synthetic portions of a humanized antibody often come from CDRs inmurine antibodies. In any event, these regions are crucial to allow theantibody to recognize and bind to a specific antigen.

As noted, murine antibodies play an important role in thesetechnologies. While useful for diagnostics and short-term therapies,murine antibodies cannot be administered to people long-term withoutincreasing the risk of a deleterious immunogenic response. Thisresponse, called Human Anti-Mouse Antibody (HAMA), occurs when a humanimmune system recognizes the murine antibody as foreign and attacks it.A HAMA response can cause toxic shock or even death.

Chimeric and humanized antibodies reduce the likelihood of a HAMAresponse by minimizing the nonhuman portions of administered antibodies.Furthermore, chimeric and humanized antibodies have the additionalbenefit of activating secondary human immune responses, such as antibodydependent cellular cytotoxicity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multi-specific antibodies formed from antibodyfragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (CL) and heavy chainconstant domains, CH₁, CH₂ and CH₃. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof.

The intact antibody may have one or more “effector functions” whichrefer to those biological activities attributable to the Fc region (anative sequence Fc region or amino acid sequence variant Fc region) ofan antibody. Examples of antibody effector functions include Clqbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

When used in vivo for combination therapy, antibodies can beadministered to the patient in therapeutically effective amounts (i.e.amounts that eliminate or reduce the patient's tumor burden). Thecombination of a compound of the disclosure and antibodies can beadministered at the same time or sequentially. They will normally beadministered parenterally, when possible, at the target cell site, orintravenously. The dose and dosage regimen will depend upon, forexample, the nature of the cancer (primary or metastatic), itspopulation, the site to which the antibodies are to be directed, thecharacteristics of the particular immunotoxin (when used), for example,its therapeutic index, the patient, and the patient's history. Theamount of antibody administered will typically be in the range of about0.1 to about 10 mg/kg of patient weight. The amount of a compound of thedisclosure administered in combination with an antibody can typicallybe, for example, in the range of about 5 to 1,000 mg, or about 0.1 to300 mg/kg of patient body weight, and can depend on many of theabovementioned factors and considerations.

The present disclosure also contemplates using combinations of acompound of the disclosure with an anti-TRP-P8 antibody in diagnosticapplications. For diagnostic applications, the antibodies of thedisclosure typically will be labeled with a detectable moiety. Thedetectable moiety can be any one capable of producing, either directlyor indirectly, a detectable signal. For example, the detectable moietymay be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescentor chemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature, 144:945 (1962); David, et al., Biochemistry,13:1014 (1974); Pain, et al., J. Immunol. Meth., 40:219 (1981); andNygren, J. Histochem. and Cytochem, 30:407 (1982).

For therapeutic applications, antibodies may be administered to amammal, preferably a human, in a pharmaceutically acceptable dosageform, including those that may be administered to a human intravenouslyas a bolus or by continuous infusion over a period of time, byintramuscular, subcutaneous, intra-articular, or inhalation routes. Anantibody is also suitably administered by intra-tumoral, peritumoral,intra-lesional, or peri-lesional routes, to exert local as well assystemic therapeutic effects.

Such dosage forms encompass known pharmaceutically acceptable carriersor vehicles that are inherently nontoxic and non-therapeutic. Anantibody will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml.

For the prevention or treatment of disease, the appropriate dosage of acombination of a compound of the disclosure will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the compound is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the compound, and the discretion of the attendingphysician. The compound is suitably administered to the patient at onetime or over a series of treatments.

Depending on the type and severity of the disease, about 0.015 to 15mg/kg of the compound is an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful.

For oral therapeutic administration, the active compound of thedisclosure may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations typically contain at least about 0.1% of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar, and the like. A syrup or elixirmay contain the active compound, sucrose or fructose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoringsuch as cherry or orange flavor. Of course, any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

The compounds or compositions of the disclosure can also be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the active compound or its salts can be prepared in water, optionallymixed with a nontoxic surfactant. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, triacetin, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes or implantableseeds or pellets. In all cases, the ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum-drying and freeze-drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. Useful dosages of thecompounds of the disclosure can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1,000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator.

For internal administration, the compositions can be administered orallyor parenterally at dose levels, calculated as the free base, of about0.1 to 300 mg/kg, preferably 1.0 to 30 mg/kg of mammal body weight, andcan be used in man in a unit dosage form, administered one to four timesdaily in the amount of 1 to 1,000 mg per unit dose.

For parenteral administration or for administration as drops, as for eyetreatments, the compounds are presented in aqueous solution in aconcentration of from about 0.1 to about 10%, more preferably about 0.1to about 7%. The solution may contain other ingredients, such asemulsifiers, antioxidants or buffers.

Generally, the concentration of the compound(s) of formula (I-XIII) in aliquid composition, such as an IV (intravenous), will be from about 0.1to about 25, preferably from about 0.5 to about 10, weight percent. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1 to about 5 weight percent, preferably about 0.5to about 2.5 weight percent.

The exact regimen for administration of the compounds and compositionsdisclosed herein will necessarily be dependent upon the needs of theindividual subject being treated, the type of treatment and, of course,the judgment of the attending practitioner.

The binding activity and binding selectivity of the compounds of thepresent disclosure are excellent predictors of the anti-tumor activityof compounds of the disclosure. The binding activity and bindingselectivity can be determined using pharmacological models well known tothe art, or using the assays described below. Exemplary results ofbiological testing are summarized in Table 1 below.

Evaluation of Biological Activity

General methods and materials disclosed in R. Skyryma, et al., J.Physiology, 2000, 527.1, 71-83, for assessing and measuring calcium ionflux or cell uptake, and induction of apoptosis were adapted for use inthe present disclosure and as illustrated herein. Other test methods andprocedures such as described below, including cell line culturing,transfection, and in vivo and in vitro tumor growth inhibition, arereadily apparent to one of ordinary skill in the art upon comprehendingthe disclosure.

Assays for Activity

For cancer, a variety of well-known animal models can be used to furtherunderstand the role of the genes in the development and pathogenesis oftumors, and to test the efficacy of candidate therapeutic agents,including combinations of compounds of the disclosure andanti-angiogenic antibodies. The in vivo nature of such models makes themparticularly predictive of responses in human patients. Animal models oftumors and cancers (e.g., breast cancer, colon cancer, prostate cancer,lung cancer, etc.) include both non-recombinant and recombinant(transgenic) animals. Non-recombinant animal models include, forexample, rodent, e.g., murine models. Such models can be generated byintroducing tumor cells into syngeneic mice using standard techniques,e.g., subcutaneous injection, tail vein injection, spleen implantation,intraperitoneal implantation, implantation under the renal capsule, ororthopin implantation, e.g., colon cancer cells implanted in colonictissue. See for example, PCT publication No. WO 97/33551, published Sep.18, 1997.

Probably the most often used animal species in oncological studies areimmunodeficient mice and, in particular, nude mice. The observation thatthe nude mouse with thymic hypo/aplasia could successfully act as a hostfor human tumor xenografts has lead to its widespread use for thispurpose. The autosomal recessive nu gene has been introduced into a verylarge number of distinct congenic strains of nude mouse, including, forexample, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA,DDD, 1/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and SJL. Inaddition, a wide variety of other animals with inherited immunologicaldefects other than the nude mouse have been bred and used as recipientsof tumor xenografts. For further details see for example, The Nude Mousein Oncology Research, E. Boven and B. Winograd, eds. (CRC Press, Inc.,1991).

The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as any of the above-listed tumor celllines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); or a moderately well-differentiated gradeII human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38); or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions involvingfreezing and storing in liquid nitrogen. Karmali et al., Br. J. Cancer,48: 689-696 (1983). Tumor cells can be introduced into animals such asnude mice by a variety of procedures. The subcutaneous (s.c.) space inmice is very suitable for tumor implantation. Tumors can be transplanteds.c. as solid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid-block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue.

Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogene wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin, et al., Proc. Nat. Acad. Sci. USA,83: 9129-9133 (1986).

Similarly, animal models of colon cancer can be generated by passagingcolon cancer cells in animals, e.g., nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, byWang, et al., Cancer Research, 54: 4726-4728 (1994) and Too, et al.,Cancer Research, 55: 681-684 (1995). This model is based on theso-called “METAMOUSE”™ sold by AntiCancer, Inc. (San Diego, Calif.).

Tumors that arise in animals can be removed and cultured in vitro. Cellsfrom the in vitro cultures can then be passaged to animals. Such tumorscan serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines. The following examples serve to more fully describethe manner of using the above-described disclosure, as well as to setforth the best modes contemplated for carrying out various aspects ofthe disclosure. It is understood that these examples in no way serve tolimit the true scope of this disclosure, but rather are presented forillustrative purposes.

Example 1

Preparation of Compound IIa-4-1 from (−)Menthyl chloride. 1.39 grams ofMg metal (57 mmol) was placed in a 100 mL flask and 4 mL of dry THF wasadded to cover the Mg metal. A crystal of iodine was added to themetal-THF mixture and stirred for several minutes followed by theaddition of about ⅓ portion of 10 grams (57 mmol) of (−) menthylchloride (Aldrich). The mixture was heated to induce Grignard formationand the remaining menthyl chloride (⅔ portion) was added in 50 mL dryTHF and stirred until the reaction was complete. The Grignard solutionwas then canulated under nitrogen into a vessel containing excess dryice. This dry ice quenched mixture was swirled and poured into 300 mLice containing 2 mL concentrated HCl. Diethyl ether was added and themixture separated. The separated organic layers were combined and washedwith water then extracted with an aqueous NaOH solution. The aqueoussolution (and oil) was acidified with HCl until a solid formed, diethylether was added, and the acid was extracted into the organic phase,washed with brine, dried over sodium sulfate, and concentrated to give4.18 grams (40%) as white needles.

The acid (0.57 grams, 3.1 mmol) was dissolved in 1.5 mLdimethylacetamide (DMA). The PyBOP (2.1 grams, 4.0 mmol), p-anisidine(0.58 grams, 4.7 mmol, Aldrich) and DiPEA (2.7 mL, 15.5 mmol) wereadded. Another 0.25 grams p-anisidine was added after 2 hours and againafter 5 hours. The reaction was diluted with ethyl acetate (EtOAc) after7 hours, washed twice with 1 N HCl, twice with aqueous sodiumbicarbonate, and then with brine. The separated organic layers werecombined and dried over sodium sulfate and concentrated to a brownsolid. This solid was put through a plug of silica gel with 30%EtOAc/hexanes to remove the color. The product began to crystallize uponconcentration, so the crystallization was allowed to occur, the solventwas removed by pipette, and the white needles were washed with coldEtOAc and dried under vacuum to give 0.365 grams (1.26 mmol, 40% yield)of a first crop. Recrystallization of the mother liquor yielded anadditional 0.346 grams (1.2 mmol, 39% yield) as white needles.

LCMS showed the product to have a molecular weight of 289, correspondingto the desired molecular weight, and the NMR spectra showed it to havethe desired structure.

Example 2

Demonstration of Ion Channel Activity in Response to Selected Cool-GenicCompounds—Calcium Ion Uptake Evaluation. Calcium ion uptake experimentswere conducted as follows. Calcium uptake was measured for cellsexpressing Trp-p8 tumor antigen and for cells not expressing Trp-p8tumor antigen after each cell line was contacted with cool-geniccompounds of the present disclosure. The results showed that cellsexpressing Trp-p8 tumor antigen had progressively increased calciumuptake as the concentration of cool-genic compound was increasedincrementally over about five concentration decades from about 0.0001 toabout 10 microM. Compound (IIa-4-1) had unexpectedly particularly highcalcium uptake at all cool-genic compound concentrations. Cells notexpressing tumor antigen had essentially no observable calcium uptakeover all of the cool-genic compound concentrations. Dimethyl sulfoxide(DMSO), a non-coolgenic compound, was used as a control compound in bothexpressing and non-expressing cell lines and which DMSO exposed cellsshowed no appreciable calcium uptake at any concentration. Cell linesincluded non-expressing lines: 293, PC3, and PC3/neo; and tumor antigenexpressing lines: 293, PC3, and PC3/neo.

Example 3

Human Cell Killing in vitro by Compounds that Activate Trp-p8. Referringto the Figures, in FIG. 1 the effectiveness of selected cool-geniccompounds in human cell killing was evaluated. The order and potency ofcool-genic compounds were compared in two related human cell lines, onewas 293 cells expressing Trp-p8 and a second was matched 293 cellslacking (not expressing) Trp-p8 used as a control. There were about50,000 cells per well at plating. The cool-genic compounds were added atplating. The cells were treated by exposing the cells to the individualcompounds at varying concentrations over about 0.1 to about 1,000 microMfor 72 hours. The x-axis shows concentration increments and the y-axisshows the number (in arbitrary units) of viable cells remaining at theend of 72 hours. FIGS. 1A and 1C show the response of cells expressingTrp-p8 and treated with the indicated numbered or named coolgeniccompounds. FIGS. 1B and 1D show the response of cells without (notexpressing) Trp-p8 and also treated with the indicated coolgeniccompounds. The results in FIGS. 1A and 1C show that the cells withTrp-p8 had a range of kill responses or potencies with respect to thecoolgenic compounds. Compound IIa-4-1 had the best kill (IC₅₀<1 microM)for cells expressing Trp-p8. The comparative results in FIGS. 1B and 1Dshow that the cells without Trp-p8 had a lower or essentially no killresponse to the same coolgenic compounds, particularly at the lowerconcentrations of the coolgenic compounds, e.g., below 100 microMolar.

Example 4

Procedure for inhibition of tumor growth in vivo by compounds thatactivate Trp-p8. Methods for preparing transfected cancerous cells, suchas those expressing Trp-p8, is readily apparent to one of ordinary skillin the art, see for example J. M. Schallhorn, et al., Nucleic AcidsRes., 1996, Feb. 15; 24(4):596-601. Referring to the FIG. 2, there isillustrated the growth of PC3 clone cells transfected to express tumorantigen, (PC3/Trp-p8.c8 and .c9), and PC3 clone cells not expressingtumor antigen (PC3-Neo) in athymic nude mice (5 million cells/mouse).The non-Trp-p8 expressing tumor forming human prostate cancer cell line,PC3-Neo 200 (--), shows expected exponential tumor cell growth rates.In contrast, the Trp-p8 expressing (i.e. “over-expressing”) tumorforming human prostate cancer cell lines, PC3/Trp-p8: .c8 (clone 8) 220(-▪-); and .c9 (clone 9) 230 (-▴-), show substantially slower growth andstatic growth rates, respectively.

Athymic mice (8 per group) are inoculated in the flank with about 5×10⁶of PC3 clone cells stablely expressing Trp-p8 (c1.8) or a vector controlcell line (PC3-Neo). Tumors are established to a size of approximately200 mg each at which time coolgenic compounds are administered once ortwice per day I.V. or P.O. at doses ranging from about 1-30 mg/kg bodyweight. Tumor volumes are measured by caliper every third day andaverage volumes are calculated.

Contacting, in vivo, the Trp-p8 expressing cancerous cells, such asthose illustrated above, with certain coolgenic compounds of moderate tohigh potency of the present disclosure, and as illustrated herein, isexpected to result in high cell kill for Trp-p8 expressing cancerouscells and no cell kill or low cell kill for healthy or non-Trp-p8expressing cells. This expectation is consistent with the abovementionedin vitro human cell killing results presented in EXAMPLE 3.

Table 1 summarizes exemplary ion channel activity and Cell Kill responseto selected cool-genic compounds, mentioned above or illustrated below,and provides a comparative or relative activity ranking for thosecompounds.

TABLE 1 Ion Channel Activity and Cell Killing in Response to SelectedCool-Genic Compounds. Activity Compound 293/Trp-p8 PC3/Trp-p8 RankingID# Peak (FLIPR)¹ Cell Killing Cell Killing 1 IIa4-1 31,000 Yes Yes 2IV-1 31,000 Yes 3 XIII-1 29,000 Yes Yes 4 IV-2 29,000 5 IIb-2 25,100 YesYes 6 IIb-3 25,100 Yes Yes 7 IIb-4 25,100 Yes 8 IIb-1 25,000 9 XIII-225,000 10 IIb-5 23,300 11 IV-3 22,000 12 Icilin 20,000 13 V-4 17,500 14V-2 17,000 15 V-1 9,600 16 III-1 5,000 17 DMSO 1,400 control ¹Peak(FLIPR) is a measure of maximum calcium ion flux at 10 microM. FLIPRrefers to Fluorometric Imaging Plate Reader; commercially availablefrom, for example, Molecular Devices Corp.

Example 5

Preparation of Other Selected Compounds of Formula IIa. Example I wasgenerally repeated for each of the following preparative compoundexamples with the exception that the amine (p-anisidine) co-reactant ofExample I was substituted with the corresponding amine to producecompound having the structure indicated as the major product uponpurification. The major product for each of the following examples wascharacterized by, for example, mass spectra to have a parent peak atabout the molecular weight indicated.

Table 2 summarizes IC₅₀ results for the indicated menthane carboxamidecompounds on 293/Trp-p8.clone 18 and 293/Trp-p8.clone 10.

TABLE 2 IC₅₀ Data for Menthane Carboxamides on 293/Trp-p8.clone 18 and293/Trp-p8.clone 10. Compound IIa2 where R₁ = H and R₂ = Compound—PhR₃R₄R₅R₆R₇ is ID# 293/Trp-p8.c18 293/Trp-p8.c10 4-MeO—Ph— IIa4-10.58/0.45 0.42 Ph— IIa4-2 3.38 3.21 4-HO—Ph— IIa4-3 1.05 1.23 3-MeO—Ph—IIa10-1 3.69 3.80 4-iPr—Ph— IIa4-4 0.98 0.76 2-MeO—Ph— IIa8-1 17.5716.74 4-Cl—Ph— IIa4-5 17.57 1.99 4-F—Ph— IIa4-6 3.78 3.58 3-F, 4-MeO—Ph—IIa6-1 0.60 0.79 4-Me—Ph— IIa4-7 1.14 1.21 4-Et—Ph— IIa4-8 0.68 0.732-Me, 4-MeO—Ph— IIa12-1 0.93 1.14 3-F—Ph— IIa10-2 2.91 6.314-morpholino-Ph— IIa4-9 1.45 1.28 3-Cl, 4-Me—Ph— IIa6-2 2.57 3.104-secBu—Ph— IIa4-10 1.08 3.59 3,4-propyleneyl-Ph— IIa6-3 1.71 1.35(i.e., indanyl) 4-tBuPh— IIa4-11 1.41 1.23 4-nPrPh— IIa4-12 4.93 5.653-Me, 4-iPrPh— IIa6-4 4.37 5.05 Icilin (reference) — — 79.24

Example 6

Table 3 summarizes IC₅₀ duplicate results for the indicated menthanecarboxamide compounds on 293/Trp-p8.clone 21.

TABLE 3 IC₅₀ Data for Menthane Carboxamides on PC3/Trp-p8.clone 21 (induplicate). Compound ID# IC₅₀ Compound IIa4 where R5- is 4-MeO—Ph—IIa4-1 1.642 4-HO—Ph— IIa4-3 4.2696 4-iPr—Ph— IIa4-4 3.2082 4-secBu—Ph—IIa4-10 8.4937 4-nPr—Ph— IIa4-12 12.857 Compound IIa4 where R5 is4-MeO—Ph— IIa4-1 1.5938 4-HO—Ph— IIa4-3 3.7606 4-iPr—Ph— IIa4-4 3.10394-secBu—Ph— IIa4-10 48.613 4-nPr—Ph— IIa4-12 16.473

Example 7

Carrier articles, such as commercially available or custom manufacturedimplantable seeds or pellets, can be formulated and impregnated with oneor more compound of the present disclosure, alone or in combination withanother chemotherapeutic agent or other medicaments or excipients. Apreferred formulation can have, for example, selectable timed-releasecharacteristics for the coolgenic compound or other ingredients, forexample, for use in prostate cancer treatment. For an example ofnon-radioactive sustained release implants in therapeutic cancertreatment, such as prostate cancer, see U.S. Pat. No. 5,633,274, whichdisclosure is incorporated herein by reference in its entirety.

Example 8

The following illustrate representative pharmaceutical dosage forms,containing a compound of the disclosure (‘Compound x’), such as acompound of formula I or II, for therapeutic or prophylactic use inhumans.

(i) Tablet 1 mg/tablet ‘Compound x’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound x’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound x’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/ml) mg/ml ‘Compound x’ 1.0 Dibasic sodiumphosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0NSodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL

(v) Injection 2 (10 mg/ml) mg/ml ‘Compound x’ 10.0 Monobasic sodiumphosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.001N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound x’ 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

Example 9

Combination Therapy-Coadministration. The following illustratesrepresentative pharmaceutical dosage forms, containing a compound of thedisclosure in direct combination (admixture) with an antibody(collectively ‘Composition y’), for therapeutic or prophylactic use inhumans. Thus, for example, a compound of the disclosure, such as acompound of formula I or II, is combined with an antibody, such as ananti-VEGF antibody. The resulting combination, ‘Composition y’, issubstituted in place of ‘Compound x’ in one or more of theabove-mentioned injection formulations of EXAMPLE 8. The aboveformulations may be obtained by conventional procedures well known inthe pharmaceutical arts.

Example 10

Combination Therapy—Serial Administration. The following illustratesrepresentative pharmaceutical dosage forms, containing a compound of thedisclosure (‘Compound x’) in serial combination with an antibody(‘antibody z’), for therapeutic or prophylactic use in humans. Thus, acompound of the disclosure, such as a compound of formula I or II, isserially administered with an antibody, such as an anti-VEGF antibody.For example, the serially administered combination can include firstadministration of a compound of the disclosure (‘Compound x’) is any ofthe above mentioned formulations of EXAMPLE 7 or 8 followed by a secondinjection administration of an antibody (‘antibody z’). Alternatively,‘antibody z’ is administered first followed by the administration of‘Compound x’. The above formulations may be obtained by conventionalprocedures well known in the pharmaceutical arts.

Example 11

Methods, examples, and additional literature references for thepreparation of antibodies, and characterization of antibodies, includingantigen specificity, epitope mapping, isotyping, binding affinity, aredisclosed in the aforementioned U.S. Pat. No. 6,582,959.

All publications, patents, and patent documents are incorporated byreference herein in their entirety, as though individually incorporatedby reference. The disclosure has been described with reference tovarious specific and preferred embodiments and techniques. However, itshould be understood that many variations and modifications can be madewhile remaining within the spirit and scope of the disclosure.

The claimed invention is:
 1. A compound of the formula IIa:

wherein R¹ is H, or (C₁-C₆)alkyl; R² is phenyl or a substituted phenylof the formula (-PhR₃R₄R₅R₆R₇)

where R₃, R₄, R₆, and R₇ are each independently —H, (C₁-C₆)alkyl,(C₁-C₆)alkoxyl, or halo; R₅ is halo, (C₁-C₆)alkyl, (C₃-C₁₂)cycloalkyl,(C₁-C₆)alkoxyl, —C(═O)(C₁-C₆)alkyl or (C₁-C₇)alkanoyl; or R₅ is —NR₈R₉,where R₈ and R₉ are each independently —H, (C₁-C₆)alkyl, or R₈ and R₉together with the nitrogen to which they are attached form a morpholino,pyrrolidino, piperidino, piperzino, indolino, benzimidazolino,azetidino, aziridino, azepino, 1,4-oxazino, or thiomorpholino ring; orR₄ and R₅ together with the phenyl to which they are attached form aring having 4 to 7 atoms and the ring having from 1 to 3 unsaturations;and stereoisomeric forms, mixtures of stereoisomeric forms; or apharmaceutically acceptable salt thereof, provided that when R₃, R₄, R₆,and R₇ of -PhR₃R₄R₅R₆R₇ are —H, R₅ is other than —CH₃, —OCH₃, —OH, —F,or —NO₂; and provided that R₂ is other than 3-hydroxy-4-methyl-phenyl;and further provided that R₂ is other than 2-hydroxy-naphthyl, orpyridyl.
 2. The compound of claim 1, wherein the compound of the formula(IIa) is: 2-Isopropyl-5-methyl-cyclohexanecarboxylic acid(4-morpholin-4-yl-phenyl)-amide;2-Isopropyl-5-methyl-cyclohexanecarboxylic acid(3-chloro-4-methoxy-phenyl)-amide;2-Isopropyl-5-methyl-cyclohexanecarboxylic acid(4-sec-butyl-phenyl)-amide; 2-Isopropyl-5-methyl-cyclohexanecarboxylicacid (4-tert-butyl-phenyl)-amide;2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (4-propyl-phenyl)-amide;and 2-Isopropyl-5-methyl-cyclohexanecarboxylic acid(4-isopropyl-3-methyl-phenyl)-amide, or pharmaceutically acceptablesalts thereof.
 3. A pharmaceutical composition comprising the compoundof claim 1 and a pharmaceutically acceptable excipient.
 4. Thecomposition of claim 3, further comprising an effective amount of atleast one additional chemotherapeutic agent for treating or preventing adisease or Trp-p8 receptor related condition.
 5. The composition ofclaim 4, wherein the chemotherapeutic agent is an antibody that causesapoptosis, inhibits angiogenesis, or both.
 6. The composition of claim5, wherein the chemotherapeutic agent is an anti-VEGF antibody.
 7. Amethod of treating cancer comprising contacting the cancer cellscomprising a Trp-p-8 receptor with an effective amount of a compound ofclaim
 1. 8. A method of treating cancer comprising contacting the cancercells comprising a Trp-p-8 receptor with an effective amount of apharmaceutical composition of claim 3.